Molecular actions of ethanol remain largely unknown. Recent developments indicate that ethanol alters intracellular signaling pathways including those involving members of mitogen activated protein (MAP) kinase family. Based on new supporting data, it is hypothesized that "ethanol modulates p38 MAPK activity leading to downstream activation of nuclear mitogen and stress activated kinase-1 (MSK-1) and phosphorylation of histone-3 as target." An important focus of this renewal application is the ethanol effects on signaling in nucleus and its consequences, e.g., on histone modifications and on apoptosis. In these studies the role of ethanol and ethanol-derived key metabolites [i.e., acetaldehyde (Acet) and phosphatidylethanol (PEth)] will be established. Four specific aims will test this hypothesis using rat hepatocytes and in vivo ethanol-fed rats as models. Aim I will determine the comparative effects of ethanol, Acet and PEth on activation/phosphorylation of p38 MAPK, MSK-1, JNK kinases. In Aim II, effects of ethanol and Acet on nuclear p38 MAPK, MSK-1 and histone-3 phosphorylation will be elucidated using pharmacological inhibitors and antisense strategy and will be correlated to apoptosis. Aim III will determine the "phospho-proteomic map" of nuclear targets affected by the above kinases through functional proteomic technique. These in vitro studies will be extended to in vivo studies in Aim IV, where chronically ethanol fed rat hepatocytes will be used to determine the modulation of p38 MAPK, MSK-1 and histone-3 phosphorylation by ethanol and Acet. The reversibility of the modulatory effects will be also investigated after ethanol withdrawal. Our focus onto nuclear effects of ethanol and its metabolites on specific members of MAPK family highlights a new dimension in ethanol research and will offer novel insight into the molecular mechanisms of ethanol actions at the subcellular organelle level. Histone modifications by ethanol will have mechanistic involvement in chromatin remodeling/gene expression. Relevance of these observations to apoptotic changes will also be established. New knowledge gained from this project will facilitate development of therapeutic tools to prevent and control ethanol-induced cellular damage.